The present invention is directed to a method for detecting methylated CpG islands. The method exploits the presence of genomic DNA sequences that exhibit altered CpG methylation patterns in many diseases, including cancer. Thus, the method is useful in the diagnosis and prognosis of such diseases.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Methylation of DNA at CpG dinucleotides is one of the most important epigenetic modifications in mammalian cells. Short regions of DNA in which the frequency of 5′-CG-3′ (CpG) dinucleotides are higher than in other regions of the genome are called CpG islands (Bird, 1986). CpG islands often harbor the promoters of genes and play a pivotal role in the control of gene expression. In normal tissue, CpG islands are usually unmethylated but a subset of islands becomes methylated during tumor development (Jones and Baylin, 2002; Costello et al., 2000; Esteller et al., 2001). Methyl-CpG binding domain (MBD) proteins specifically recognize methylated DNA sequences and are essential components of regulatory complexes that mediate transcriptional repression of methylated DNA (Hendrich and Bird, 2000; Wade, 2001). One of the best-characterized members of the MBD protein family is MBD2. MBD2 has two isoforms, MBD2a and MBD2b, which are alternatively translated from the same mRNA (Hendrich and Bird, 1998). Recent studies indicate that interacting proteins can modulate the methylated DNA-binding ability of the MBD2 protein (Jiang et al., 2004). MBD3L1 interacts with MBD2b in vivo and in vitro and promotes the formation of larger methylated-DNA binding complexes (Jiang et al., 2004).
In order to identify and characterize the chromosomal regions (particularly CpG islands) that undergo de novo methyation in tumorigenesis many technical approaches have been used (Costello et al., 2000; Fraga and Esteller, 2002; Shiraishi et al., 2002a). These methods can be classified into several groups on the basis of their principles. The first group of techniques is based on restriction endonuclease cleavage. These techniques require the presence of methylated cytosine residues at the recognition sequence that affect the cleavage activity of isoschizomeric restriction endonucleases (e.g., HpaII and MspI) (Singer et al., 1979). In this method the methylation sensitive and resistant enzyme pair produces characteristic fragment populations of the genomic DNA that can be detected by Southern blot hybridization. The technique is limited to specific restriction sites and requires large amounts of genomic DNA.
The second set of techniques makes use of the differential sensitivity of cytosine and 5-methylcytosine towards chemical modification and cleavage by employing Maxam-Gilbert sequencing technology (Maxam and Gilbert, 1980). The application of ligation-mediated PCR techniques to Maxam-Gilbert treated genomic DNA allows the exact identification and partial quantification of 5-methylcytosines at the single nucleotide level in mammalian genes (Pfeifer et al., 1989). Although highly specific and reasonably sensitive (requires 0.5 μg to 1 μg of DNA) these techniques are technically complex. The principle of bisulfite genomic sequencing is that methylated and unmethylated cytosine residues react in a different manner with sodium bisulfite (Clark et al., 1994; Frommer et al., 1992). After bisulfite treatment of genomic DNA, the unmethylated cytosines are converted to uracils by deamination, while methylated cytosine residues can hardly react with this agent and remain intact. After this chemical treatment the region of interest must be PCR amplified, and in most cases cloned and sequenced. Alignment analysis of the original (untreated) and cloned (treated) nucleotide sequences can reveal the in vivo methylation status of the amplified region. The PCR products obtained from bisulfite-treated DNA can also be further analyzed by combined bisulfite-restriction analysis (COBRA assay), which can distinguish between methylated and unmethylated DNA (Xiong and Laird, 1997).
Another commonly used sodium bilsulfite dependent technique is methylation-specific PCR (MSP) (Herman et al., 1996). Sodium bisulfite treated genomic DNA serves as the template for a subsequent PCR reaction. Specific sets of PCR primers are designed in such a way to discriminate between bisulfite modified and unmodified template DNA and between unmethylated (deaminated) and methylated (non-deaminated) cytosines at CpG sites. Another approach used for the identification of methylated CpG islands utilizes the ability of the MBD domain of the MeCP2 protein to selectively bind to methylated DNA sequences (Cross et al., 1994; Shiraishi et al., 1999). The bacterially expressed and purified His-tagged methyl-CpG binding domain is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Restriction endonuclease digested genomic DNA is loaded onto the affinity column and methylated CpG island enriched fractions are eluted by a linear gradient of sodium chloride. PCR or Southern hybridization techniques are used to detect specific sequences in these fractions. There are several additional methods for analysis of methylation patterns but each of them is a derivative of the above-mentioned principles (Fraga and Esteller, 2002; Shiraishi et al., 2002a).
Most of the currently used methods for detecting methylated CpG islands, as described above, are based on sodium bisulfite conversion of genomic DNA followed by PCR reactions. In addition, most methods currently available are labor-intensive and use methylation-sensitive restriction endonucleases and thus are limited by the occurrence of the respective sites within the target sequence.
In addition to the above techniques, another way to find methylated genes is by using expression microarrays to identify genes reactivated by treatment with DNA methylation inhibitors, e.g. 5-aza-deoxycytidine (Shi et al., 2003; Suzuki et al., 2002; Yamashita et al., 2002). This approach can only be used with cell lines. Recently, genomic tiling and BAC microarrays have been introduced to map methylation patterns (Ching et al., 2005; Weber et al., 2005). These approaches are also limited, both in terms of their level of resolution and in terms of the requirements for restriction endonuclease recognition sites. An antibody against 5-methylcytosine has been used in immunoprecipitation experiments combined with microarrays (Weber et al., 2005; Keshet et al., 2006). However, this antibody requires single-stranded DNA for recognition, which is often difficult to achieve in CpG-rich regions.
In view of the above described disadvantages, it is desired to develop a methylation assay that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches and is less laborious. Such a methylation assay would be clinically useful in the early detection and diagnosis of any DNA methylation related disease, including cancer. It is also desired to adapt this methylation assay to microarray analysis for the determination of genome-wide DNA methylation patterns.